Whole-genome characterisation of TEM-1 and CMY-2 β-lactamase-producing Salmonella Kentucky ST198 in Lebanese broiler chain

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To cite this version:

Hage, Rima el

and Losasso, Carmen and Longo, Alessandra and Petrin, Sara

and Ricci, Antonia and Mathieu, Florence

and Abi Khattar, Ziad and El Rayess,

Youssef Whole-genome characterisation of TEM-1 and CMY-2

β-lactamase-producing Salmonella Kentucky ST198 in Lebanese broiler chain. (2020) Journal

of Global Antimicrobial Resistance, 23. 408-416. ISSN 2213-7165

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Whole-genome

characterisation

of

TEM-1

and

CMY-2

β-lactamase-producing

Salmonella

Kentucky

ST198

in

Lebanese

broiler

chain

Rima

El

Hage

a,d,

*

,

Carmen

Losasso

c

,

Alessandra

Longo

c

,

Sara

Petrin

c

,

Antonia

Ricci

c

,

Florence

Mathieu

d

,

Ziad

Abi

Khattar

b,

**

,

Youssef

El

Rayess

e,

**

a

LebaneseAgriculturalResearchInstitute(LARI),FanarStation,FoodMicrobiologyLaboratory,JdeidehEl-Metn,Lebanon

b

LebaneseUniversity,FacultyofSciences2,L2GE,Microbiology-Tox/EcotoxTeam,Fanar,Lebanon

c

IstitutoZooproﬁlatticoSperimentaledelleVenezie,Legnaro(PD),Italy

d

UniversitédeToulouse,LaboratoiredeGénieChimique,UMR5503CNRS/INPT/UPS,INP-ENSAT,1Avenuedel’Agrobiopôle,31326Castanet-Tolosan,France

e

HolySpiritUniversityofKaslik,FacultyofAgriculturalandFoodSciences,Jounieh,Lebanon ARTICLE INFO

Articlehistory: Received2August2019

Receivedinrevisedform25September2020 Accepted2November2020

Availableonline16November2020 Keywords:

Lebanon Poultry

Ciproﬂoxacin-resistantSalmonellaKentucky ST198 blaCMY-2 IS10transposition ISEcp1 Plasmids ABSTRACT

Objectives:Salmonellaentericasubsp.entericaserovarKentuckyhasbeenassociatedwiththeworldwide ciproﬂoxacin-resistant(CIPR₎_Salmonella_Kentucky_sequence_type₁₉₈_(ST198)_epidemic_clone,_mostly recoveredfrompoultryfarmsandproducts.Theaimofthisstudywastoexaminewhetherthisexpanding cloneexistsintheLebanesebroilerchain.

Methods: Eight CIPR _and _{extended-spectrum} _{cephalosporin-resistant} _Salmonella _Kentucky _isolates previously recovered from Lebanese broilers were genetically characterised by whole-genome sequencing.

Results:SevenoftheeightisolatesbelongedtoST198andwerephylogeneticallycloselyrelated.Theyall harbouredmutationsinthechromosomalquinoloneresistancegenesgyrAandparCwithdoubleand singlesubstitutions,respectively.TheblaTEM-1BandblaCMY-2geneswerebothdetectedinsixisolates. InsertionsequenceISEcp1waslocatedupstreamofblaCMY-2,harbouredbyIncI1plasmidsinfourstrains. AnIS10transpositioncoupledtohomologousrecombinationattranspositionsites mediatedCMY-2 plasmidintegrationintothechromosomeofonestrain.Resistancegenestoaminoglycosides[aadA7and aac(3)-Id],tetracyclines[tet(A)]andsulfonamides(sul1)weredetectedinﬁvestrains,amongwhichfour werepositiveforthepresenceofSalmonellagenomicisland1(SGI1)variantSGI1-K.Allstudiedisolates harboureda varietyof Salmonella pathogenicity islands(SPIs) as wellas commonregulatory and virulencegenes.

Conclusion:HerewereportfortheﬁrsttimeinLebanonthedetectionanddisseminationoftheemerging highlydrug-resistantSalmonellaKentuckyST198.Ourﬁndingsshednewlightonthiscloneasapotential public-healththreat.

1.Introduction

Uncommoninhumansalmonellosis,Salmonellaentericasubsp. entericaserovarKentuckyishoweverwidespreadinpoultrymeat

[1].FirstrecordedinEgyptin2002,anewcloneofcipro ﬂoxacin-resistant(CIPR₎_Salmonella_Kentucky_sequence _type₁₉₈ _(ST198)

hasspread worldwide[2,3],causinghumaninfectionslinkedto travellers returning from the Middle East, Southeast Asia and Africa [4]. Since the 1990s, this serovar acquired Salmonella genomicisland1(SGI1)multidrugresistancedeterminantsmainly toamoxicillin, gentamicin and sulfonamides as wellas double mutationsintopoisomerase-encodinggyrAandparCchromosomal genesconferringresistancetociproﬂoxacin[5].

Mediterranean Salmonella Kentucky isolates have become

producersofvariouscarbapenemases(blaVIM-2,blaOXA-48,bla OXA-204),extended-spectrumβ-lactamases(ESBLs)(blaCTX-M-1,bla CTX-M-15,blaCTX-M-25)andamixofcarbapenemasesandESBLs(blaOXA-48

and blaVEB-8), posing an imminent threat to public health [6]. *Correspondingauthorat:LebaneseAgriculturalResearchInstitute(LARI),Fanar

Station,FoodMicrobiologyLaboratory,JdeidehEl-Metn,Lebanon. **Correspondingauthors.

E-mailaddresses:relhage@lari.gov.lb(R.ElHage),ziad.abikhattar@ul.edu.lb (Z.AbiKhattar),youssefrayess@usek.edu.lb(Y.ElRayess).

http://dx.doi.org/10.1016/j.jgar.2020.11.002

ContentslistsavailableatScienceDirect

Journal

of

Global

Antimicrobial

Resistance

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Anotherclassofβ-lactamases,theAmpCβ-lactamases,which is furtherdivided intodifferentfamilies (CIT, CMY,FOX,etc.) and types or variants (CMY-2, DHA-1, FOX-1, etc.), has emerged worldwideasclinicallyrelevantinEnterobacteriaceae.Theyconfer resistancetoaminopenicillins,cephalosporins(ceftriaxone, cefo-taxime, ceftazidime), cephamycins (cefoxitin, cefotetan) and monobactams(aztreonam)[7].AccordingtotheAmblerstructural classiﬁcation,AmpCβ-lactamasesbelongtothemolecularclassC β-lactamases originally described as inducible chromosomal enzymes [8].blaCMY-2 is themost prevalent AmpCβ-lactamase

gene,which hasbeenreportedinS.enterica andEscherichiacoli worldwideowingtothespreadofIncA/CandIncI1plasmidsfrom differentsourcesincludinghumans,animalsandtheenvironment

[7].Theplasmid-borneblaCMY-2genemostlikelyoriginatedfrom

theCitrobacterfreundiichromosomebyinsertionsequence ISEcp1-mediated transpositionthat alsoprovides thepromotor for its high-level expression[9].AmpCβ-lactamaseproduction canbe assessedusingthecefoxitindiskscreeningtest[8]aswellasthe cefoxitin/cloxacillin double-disk and AmpCinduction tests that allowdetectionofAmpCβ-lactamaseproductionin Enterobacter-iaceaenaturallylackingchromosomalAmpCβ-lactamases,suchas Salmonellaspp.MultiplexPCRassayshavealsobeendescribedfor speciﬁc detection of six families of plasmid-acquired AmpC β-lactamasegenes[10].

Some studies have shown that multidrug-resistant

(MDR) Salmonella and ESBL-producing isolates became more

pathogenicby co-carryingseveral virulence genesonplasmids, prophages and Salmonella pathogenicity islands (SPIs) [11]. Some virulence genes were identiﬁed to confer pathogenicity morethanothers.SalmonellaKentuckyisthoughttobeunharmful

to humans owing to the lack of many virulence genes such

asgrvA,sseI,sopEandsodC1[12],orsopD2,pipB2,sspH2andsrfH

[13]. The relevant concern with Salmonella Kentucky is its accelerateddisseminationinchickensattributedtoabetteracid responsethanotherserovars[14].Othersattributedthe differen-tialregulationofcoreSalmonellagenesviathestationary-phase sigma factor RpoS tothe metabolic adaptation in the chicken caecum[12].

InLebanon,SalmonellaKentuckyisamongthemost predomi-nant serovars in the broiler production chain (broiler breeder farms,broilerfarms,slaughterhousesandretail)andlayerﬂocks. Theglobalprevalenceofthisserovarwas21.4%amongthetotal identiﬁedones(unpublishedresults),althoughitwasnotrelated tohumaningestion[15].Ithasbeenshownthatallisolatedstrains wereCIPR_,_65.4%_were_MDR_and_6.8%_were_also_{extended-spectrum}

cephalosporin-resistant(ESCR_)._The_aim_of_this_study_was_therefore

todeterminewhethertheseCIPRand ESCRSalmonellaKentucky strainsbelongedtotheexpandingST198-SGI1clone.Inlinewith this,adeepgenomiccharacterisationwasperformed.

2.Materialsandmethods

2.1.CollectionofSalmonellaKentuckystrains

Eight Salmonella Kentucky isolates were chosen from a

collectionofstrainsisolatedduringthesameyearinourlaboratory asapartofapreviousstudyonSalmonellaprevalenceinLebanese poultry production. Of a total 133 CIPR _Salmonella _Kentucky

isolates,only8wereselectedforwhole-genomesequencing(WGS) accordingtotheirESCRphenotypicproﬁle.Thecollectedstrains originated as follows: seven strains [17-70328(K12), 17-70460 (K24),17-70462(K31), 17-70464(K32),17-70468(K38), 17-70469 (K43)and17-70472(K48)]fromretailchickencutsandonestrain [17-70474(A66C)] from commercialslaughterhouse broiler cae-cum(unpublishedresults).

2.2.Antimicrobialsusceptibilitytesting

Antimicrobialsusceptibilitytestingwasperformed accord-ing tothe Clinical and Laboratory Standards Institute(CLSI)

[16]. The Kirby–Bauer disk diffusion method was ﬁrst

performed for a panel of 26 antimicrobials (Oxoid Ltd.,

Basingstoke,UK)ofveterinaryandhumanhealthimportance, including ampicillin (10

m

g), amoxicillin/clavulanic acid (30

m

g), piperacillin/tazobactam (110

m

g), cefalotin (30

m

g), cefuroxime (30

m

g), cefoxitin (30

m

g), cefotaxime (30

m

g), ceftriaxone (30

m

g), ceftazidime (30

m

g), ceftiofur (30

m

g),

cefepime (30

m

g), imipenem (10

m

g), aztreonam (30

m

g),

gentamicin(10

m

g),tobramycin(10

m

g),streptomycin(10

m

g), amikacin(30

m

g), netilmicin(30

m

g), nalidixicacid(30

m

g), ciproﬂoxacin(5

m

g),norﬂoxacin(10

m

g),enroﬂoxacin(5

m

g), trimethoprim (5

m

g), trimethoprim/sulfamethoxazole (1.25/ 23.75

m

g),tetracycline(30

m

g)andchloramphenicol(30

m

g). Minimuminhibitory concentrations (MICs)forresistantstrainswere determinedbybrothmicrodilutionforthefollowingantimicrobials (breakpoint values in parentheses): cefalotin (32

m

g/mL); cefuroxime (32

m

g/mL); cefoxitin (32

m

g/mL); cefotaxime (4

m

g/mL);ceftriaxone (4

m

g/mL); ceftazidime(16

m

g/mL); ceftiofur (8

m

g/mL); gentamicin (16

m

g/mL); nalidixic acid (32

m

g/mL);ciproﬂoxacin(1

m

g/mL);norﬂoxacin(16

m

g/mL); andenroﬂoxacin(2

m

g/mL).EscherichiacoliATCC25922wasused asaqualitycontrolstrain.Antimicrobialresistancetoatleastthree classesofantibioticswasconsideredMDR.

2.3.Whole-genomesequencinganalysis

GenomicDNAwasextractedusingaQIAamp1DNAMiniKit (QIAGEN,Valencia,CA,USA)andwasquantiﬁedwithaQubit

3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA).

Libraries for sequencing were prepared using a Nextera XT

DNALibraryPreparationKit(IlluminaInc.,SanDiego,CA,USA).

High-throughput sequencing was performed on an Illumina

MiSeq system (Illumina Inc.) with 2 250-bp paired-end

reads. Raw sequence data were submitted to the European

Nucleotide Archive (http://www.ebi.ac.uk/ena) under acces-sionno.PRJEB27597.Rawreadswereassembledincontigsusing Assembler1.2(https://cge.cbs.dtu.dk/services/Assembler/)[17]or SPAdes3.9(https://cge.cbs.dtu.dk/services/SPAdes/)[18].All

iso-lates were then subjected to in silico serotyping using

SeqSero 1.2 (www.denglab.info/SeqSero) [19] starting from assembleddatatoconﬁrminvitroserotyping.Whenconcordance wasnotveriﬁed,analysiswasrepeatedstartingfromrawreads.To verify the presence of acquiredantimicrobial resistance genes,

assembled genomes were analysed using ResFinder2.1

(https://cge.cbs.dtu.dk/services/ResFinder/) (selected threshold

for %ID = 90%; selected minimum length = 60%), while

Res-Finder3.0 (https://cge.cbs.dtu.dk/services/ResFinder-3.0/) [20]

was used to detectknown chromosomal point mutations that

canconfer antimicrobialresistance. Multilocussequence typing (MLST),plasmididentiﬁcationandplasmidMLST(pMLST)were performedusingMLST1.8(https://cge.cbs.dtu.dk/services/MLST/)

[17], PlasmidFinder 1.3 ( https://cge.cbs.dtu.dk/services/Plasmid-Finder/)(selectedthresholdfor%ID=85%)andpMLST1.4(https:// cge.cbs.dtu.dk/services/pMLST/) [21], respectively. MyDbFinder (https://cge.cbs.dtu.dk/services/MyDbFinder/) was used to

investigate the presence of SGI1-K (GenBank accession no.

AY463797.8) [22], which is frequently integrated into the SalmonellaKentuckygenome.Thereferenceusedto_ﬁndISEcp1 was the deposited sequence of Salmonella Typhimurium strain 110516[KX377449.1:780–1276].

R.ElHage,C.Losasso,A.Longoetal./JournalofGlobalAntimicrobialResistance23(2020)408–416

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2.4.Phylogenomics

Assembled genomes and a reference genome (Salmonella

KentuckyCVM29188[23])wereusedtobuildasinglenucleotide polymorphism(SNP)-basedphylogenetictreeusingCSIPhylogeny 1.4 (https://cge.cbs.dtu.dk/services/CSIPhylogeny/)[24],with de-faultparametersforSNPﬁlteringandSNPpruning.

3.Results

3.1.MLST,plasmiddetectionandpMLST

AllisolatessubmittedtoWGSbelongedtoST198,exceptfor 17-70472(K48)forwhichitwasnotpossibletoassignaMLST

sequence type. This was most likely due to a bad assembly

comparedwithotherisolates,since17-70472(K48)showed a high number of contigs (3495 contigs) and a low N50value

(Table1).

Using WGS data, all plasmids recovered from all isolates belongedtoincompatibilitygroupI1(IncI1)andColRNAIreplicon types.UsingpMLSTbasedonWGSdata,twoIncI1-typeplasmids wereST12andtwootherplasmidswereidentiﬁedasbelongingto ST2andST65.Thefourotherswereuntypeable,buttwoofthem closelymatchedST12andST23(Table1).

3.2.Phenotypicandgenotypicantimicrobialresistance

Antimicrobial susceptibility testing showed high MICs to ciproﬂoxacin(12.5

m

g/mLto>32

m

g/mL) forallstrains, among which six were classiﬁed as MDR. Phenotypic antimicrobial resistancepatternsarereportedinTable2.

WGS data revealed that all of the isolates had all genes correlatingwiththe antimicrobialresistance phenotypes deter-minedbydiskdiffusionandbrothmicrodilutionmethods.Indeed, ESCR _strains _were _found _to _carry _resistance _genes _to

third-generation β-lactams, blaTEM-1B (class A) and/or cephamycinase

blaCMY-2(classC),withsixstrainscarryingbothofthem(Table2).

For strain 17-70468(K38), although initial disk diffusion test revealed a resistant phenotype to cefoxitin, the blaCMY-2

resistance gene was not detected. Broth microdilution demon-strated that this strain had reduced susceptibility to cefoxitin (MIC=12.5

m

g/mL)comparedwiththeothercefoxitin-resistant strains(MIC200

m

g/mL).

Moreover,mutationsinthequinoloneresistance-determining regions (QRDRs) of the target gyrA and parC gene loci were detectedinallstrains.Thestrainsharboureddoubleaminoacid substitutionsinGyrA[serinetophenylalanineatcodon83(S83F) andasparticacidtoasparagineatcodon87(D87N)]andasingle substitutioninParC[serinetoisoleucineatcodon80(S80I)].These mutationswerethereforeresponsibleforhigh-levelciprofloxacin resistance(Table2).Resistancegenestoaminoglycosides[aadA7 andaac(3)-Id],tetracyclines[tet(A)]andsulfonamides(sul1)were detectedinfivestrains,amongwhichfourharbouredtheSGI1-K variant.Strain17-70462(K31)wastheonlystraintohavethefloR geneconferringcross-resistancetochloramphenicoland florfeni-col(Table2).

3.3.CMY-2plasmidanalysis

NucleotidesequenceanalysisrevealedthepresenceofISEcp1in ﬁvestrainsat117bpupstreamoftheblaCMY-2gene.Thelatterwas

co-localised on the same contig with both IncI1-type plasmid repliconandISEcp1.However,strain17-70460(K24)lackedISEcp1 and harbouredblaCMY-2 and IncI1 on two differentcontigs. As

showninTable3,thefourISEcp1–blaCMY-2containingcontigsfrom

strains 17-70462(K31), 17-70464(K32), 17-70469(K43) and 17-70474(A66C)displayedrelativelyshortlengths.Theyalsoshared high identities (98.59–100%) with the previously described blaCMY-2IncI1 plasmidpCVM29188_101 (GenBankaccession no.

CP001121.1)ofSalmonellaKentuckyfrompoultry[23]anditsclose

variants (p12-4374_96, CP012929.1 and pSA01AB09084001_92,

CP016533.1) in Salmonella Heidelberg recently isolated from different sources in Canada [25]. These observations indicated that the ISEcp1–blaCMY-2 transposition unit resided on IncI1

plasmids belonging to two close variants 1 and 2 (Fig. 1A), seeminglyhorizontallyspreadamongSalmonellaserovars world-wide.ISEcp1–blaCMY-2wasfollowedbyfull-lengthblc(encodingan

outer membrane lipoprotein) and sugE (encoding a quaternary

ammonium compound resistance protein) genes along with a

551-bpfragment(Fig.1A).Thisfragmentshowednullidentityto

Salmonella Kentucky sequences including the pCVM29188_101

plasmid, but was 100% identical to a region containing a

5ʹ-truncated LuxR family transcriptional regulator (ecnR) gene belongingtoIncA/Cplasmidsfromotherserovars.

For the remaining strain 17-70328(K12), the blaCMY-2 gene

residedonthelargestcontig(1758.535kb)correspondingtothe

Table1

Resultsofgenomicassembly,SeqSero,multilocussequencetyping(MLST),PlasmidFinderandplasmidMLST(pMLST)andaccessionnumberofeightLebaneseSalmonella Kentuckyisolates.

IDIZSVe(Ref. Lebanon)

Source Genomesize (bp)

No.of contigs

N50a Serovar MLST Plasmids pMLSTb Accessionno.

17-70328(K12) Chickencuts/retail 4922807 93 534 536 Kentucky ST198 IncI1, ColRNAI IncI1[ST65] ERR2681948 17-70460(K24) Chickencuts/retail 5002563 251 238 030 Kentucky ST198 IncI1, ColRNAI IncI1[ST12] ERR2681949 17-70462(K31) Chickencuts/retail 4967065 105 534 536 Kentucky ST198 IncI1, ColRNAI IncI1[ST12] ERR2681950 17-70464(K32) Chickencuts/retail 4916713 80 450 673 Kentucky ST198 IncI1, ColRNAI

IncI1[unknown,closest matchST23]

ERR2681951 17-70468(K38) Chickencuts/retail 4980697 458 26113 Kentucky ST198 IncI1,

ColRNAI

IncI1[unknown,closest matchST12] ERR2681952 17-70469(K43) Chickencuts/retail 4896047 93 293 715 Kentucky ST198 IncI1, ColRNAI

IncI1[unknown] ERR2681953 17-70472(K48) Chickencuts/retail 4417617 3495 1670 Kentucky Unknown IncI1 IncI1[unknown] ERR2681954 17-70474(A66C) Chickencaecum/

slaughterhouse 4942747 149 120 678 Kentucky ST198 IncI1, ColRNAI IncI1[ST2] ERR2681955 a

TheN50statisticdeﬁnesassemblyquality.Givenasetofcontigsorderedfromtheshortesttothelonger,N50isdeﬁnedastheshortestsequencelengthamongcontigsthat

coversatleastone-halfofthegenomesize.

b

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chromosome,suggestingthatthisplasmidmighthaveintegrated

into the chromosome. An alignment of this contig against

sequences from various online databases identiﬁed a 93.8-kb large fragment sharing the highest signiﬁcant identity (91%

coverage and 98.59% identity) with pCVM29188_101 plasmid

(Fig.1B).TheplasmidinsertionsitewasﬂankedbytwoIS10with thesameorientation[left(IS10L)andright(IS10R)]accompanied bytypicaltargetsite9-bpduplication(DR1).Theresultinggenetic structure IS10L-93.8 kb fragment-IS10R mimicked a composite transposonthathasbeeninsertedintothechromosomalhemin uptakeprotein-encodinghemPgene.Similarly,twoother9-bpDR2 weredetectednext toinvertedrepeatsIRRand IRLofIS10Land

IS10R,respectively.Geneticmappinganalysisrevealedthatsucha

genomic organisation could have resulted from homologous

recombination attheIS10 site itselfbetweentheIncI1 plasmid andthechromosome.

Sequenceanalysisofstrain17-70328(K12)alsoshowedthatthe ISEcp1–blaCMY-2 transposition unit was inserted into the

SeKA_C0024 gene at 21.4 kb upstream of IS10R, followed by theblcgeneandatruncatedformofsugE(Fig.1B).Alltypical14-bp IRsand5-bpAT-richDRs(DR3)ofISEcp1wereidentiﬁed,among whichtheIRLdetectedat20-bpupstreamofthe3ʹendoftheyagA

geneandthealternativeIRaltbarelyrecogniseddirectlyupstream

oftheSeKA_C00243ʹ-part.Thisexplainedwhytheterminal34-bp of yegA belonged to the ISEcp1 transposition unit that was extendedbeyond theIRRcausing alargerregiontobecaptured

andinsertedintotheSeKA_C0024locus.The5ʹ-partofsugEwas left in tandem with the remaining part of yagA. Many genes indicative ofmotility, includingtransposasesand recombinases, wereobservedalongwithdeletionsandinsertionsofsequences identicaltotheIncFIB/IncFIIApCVM29188_146(CP001122.1)and

IncI1 pCS0010A (CP0020901) plasmids previously found in

Salmonella Kentucky. The presence of such diverse plasmid

sequences suggeststhat plasmids of differentInc groups could havecoexistedinancientSalmonellaKentuckyhostswheretheir genes were subject to intermolecular and intramolecular rear-rangements. In line with this, we observed the presence of a ‘TGGGT’DR(typicalofIncI1pCVM29188_101)onthesugE5ʹ-endat the original deletion site of ISEcp1. This could indicate that

recombinations in this region have occurred before horizontal acquisitionoftheblaCMY-2plasmidanditsintegrationintothe

17-70328(K12)chromosome.Finally,severalgeneswerenot integrat-edintothechromosome,includingyacABC and ccdABencoding toxin–antitoxin plasmidstabilitysystems. Therefore,thelossof

these systems along with continuous exposure to increased

cephalosporin concentrations could have promoted the CMY-2 plasmidintegrationintothechromosome.

3.4.Virulencegeneanalysis

ScreeningofSPIs,virulencegenes,RpoS-regulatedcoregenesas well as other genes related to pathogenicity and survival of SalmonellaKentuckywasperformed(Fig.2).Allsequencedstrains

were shown to have conserved genes implicated in

ﬁmbriae-mediated adhesion, curli formation, iron acquisition, galactose transport, propionate catabolism, nitrate respiration, type III secretionsystem(T3SS)andregulationofstressfactors.However,

SPI-1 and SPI-2 were detected in four and three strains,

respectively, whilstonlytwo strains harbouredthem both.The SPI-1sopE2genewas absentfromtwostrains, whileallstrains lackedtheSPI-2sspH2gene.Thessek2genewasidentiﬁedinsix strainsbutwas missinginallsopE2-lackingstrains.Finally,itis

noteworthy to state that strain 17-70472(K48) lacked many

virulence genes,namelylpfD,stjB, siiE,sopE2, ssek2, pipA, pipD, mgl, prp and nar, which could impact its _ﬁtness in chicken colonisationandhumaninfections.

3.5.PhylogeneticSNPanalysis

SNPsidentiﬁedfromwhole-genomecomparisonswereusedto determinethe relationships betweenthe eight CIPRSalmonella

Kentucky strains with Salmonella Kentucky CVM29188 strain

selectedasareferencegenome.Asshownbythephylogenetictree inFig.3,acloserelatednessbetweenallstrainswasobserved,with

the exception of 17-70472(K48). Once again, this was most

probably due to the bad assembly achieved for this particular strain.SNPdifferenceamongstrainsvariedbetween12and7491 nucleotides.

Table2

Phenotypicandgenotypicantimicrobialresistance(AMR)resultsoftheeightLebaneseciproﬂoxacin-resistantSalmonellaKentuckyisolatesusingResFinder2.1,ResFinder3.0 andMyDbFinder.

IDIZSVe(Ref. Lebanon)

AMRphenotype AMRgenotype QRDRpoint

mutations

SGI1-K gyrA parC 17-70328(K12) AMP-AMC-CEF-CXM-FOX-CTX-CRO-CAZ-TIO-NAL-CIP-NOR-ENR blaCMY-2,blaTEM-1B S83F,

D87N

S80I Absence 17-70460(K24)

AMP-AMC-CEF-CXM-FOX-CTX-CRO-CAZ-TIO-GEN-STR-NAL-CIP-NOR-ATM-TET-ENR

aadA7,aac(3)-Id,blaCMY-2,blaTEM-1B,sul1,

tet(A)

S83F, D87N

S80I Absence 17-70462(K31) AMP-AMC-CEF-CXM-FOX-CTX-CRO-CAZ-TIO-NAL-CIP-NOR-CHL-ENR blaCMY-2,blaTEM-1B,ﬂoR S83F,

D87N

S80I Absence 17-70464(K32) AMP-AMC-CXM-FOX-CRO-CAZ-TIO-GEN-STR-NAL-CIP-NOR-ATM-TET-ENR aadA7,aac(3)-Id,blaCMY-2,blaTEM-1B,sul1,

tet(A) S83F, D87N S80I Presence 17-70468(K38)a AMP-AMC-CEF-CXM-FOX-CTX-CRO-CAZ-TIO-GEN-STR-NAL-CIP-NOR-TET-ENR

aadA7,aac(3)-Id,blaTEM-1B,sul1,tet(A) S83F,

D87N

S80I Presence 17-70469(K43)

AMP-AMC-CEF-CXM-FOX-CTX-CRO-CAZ-TIO-GEN-STR-NAL-CIP-NOR-ATM-TET-ENR

tet(A)

S83F, D87N

S80I Presence 17-70472(K48) AMP-AMC-CEF-CXM-FOX-CTX-CRO-CAZ-TIO-NAL-CIP-NOR-ENR blaCMY-2 S83F,

D87N

S80I Absence 17-70474(A66C) AMP-AMC-TZP-CEF-CXM-FOX-CTX-

CRO-CAZ-TIO-GEN-STR-NAL-CIP-NOR-ATM-TET-ENR

tet(A)

S83F, D87N

S80I Presence QRDR,quinoloneresistance-determiningregion;SGI1-K,Salmonellagenomicisland1variant;AMC,amoxicillin/clavulanicacid;AMP,ampicillin;ATM,aztreonam;CAZ, ceftazidime;CEF, cefalotin;CHL,chloramphenicol;CIP,ciprofloxacin;CRO,ceftriaxone;CTX,cefotaxime;CXM,cefuroxime;ENR,enrofloxacin;FOX,cefoxitin;GEN, gentamicin;NAL,nalidixicacid;NOR,norfloxacin;STR,streptomycin;TET,tetracycline;TIO,ceftiofur;TZP,piperacillin/tazobactam.

a

Categorisedasresistantaccordingtodiskdiffusionmethodandofreducedsusceptibilityaccordingtobrothmicrodilution. R.ElHage,C.Losasso,A.Longoetal./JournalofGlobalAntimicrobialResistance23(2020)408–416

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4.Discussion

In this study, for the ﬁrst time in Lebanon, MLST analysis performed on eight Salmonella Kentucky isolates from poultry showedthatsevenisolatesbelongedtotheinternationalemerging CIPRSalmonellaKentuckyST198 clone,of whichsix wereMDR. Double substitutions in GyrA (Ser83 and Asp87) and a single substitutioninParC(Ser80)arefrequentlyidentiﬁedinCIPR_strains

[4],whichwasalsothecaseforallourstrainshighlyresistantto ﬂuoroquinolones.

The blaTEM-1B and/or blaCMY-2 genes were detected in our

isolates,conﬁrmingthecurrenthypothesisthattheMediterranean Basin is the ecological niche of _{β-lactam-resistant} Salmonella KentuckyST198.Liakopoulosetal.showedthattheemergenceof ESCR _Salmonella_in _the_Netherlands_was _due _to_the_presence _of

blaCMYon IncI1 plasmids [26]. Similarly,suchan IncI1 plasmid

repliconwasfoundinallisolatesinvestigated.Moreover,plasmid

sequenceswerediversewithintheseisolates,amongwhichtwo wereidentiﬁedastheIncI1/ST12.Thelatterhasbeendisseminated worldwide,beingrelated tothespread of blaCMY-type

plasmid-mediated AmpCgenesamong Enterobacteriaceae [27].ISEcp1is ofteninsertedbytranspositionatthe5ʹendofβ-lactamasegenes providingpromotersequencesforexpression,therebyenablingan increase in MICs of ESCs such as cefotaxime, ceftiofur and ceftazidime [28]. We showed here that the highly cefoxitin-resistant phenotype was associated with the IncI1 plasmid-encodedCMY-2 locateddownstream ofISEcp1.Indeed, theonly knownβ-lactamresistancegenedetectedinstrain17-70468(K38) withreducedcefoxitinsusceptibilitywasblaTEM-1B.Nonetheless,

cefoxitinisknowntobestableagainstTEM-1activity[29].Thus,it is quite likely that the reduced cefoxitin susceptibility of this particular isolate could have resulted from other mechanisms relatedtooutermembranepermeabilitynotinvestigatedinthis study[30].

Table3

Resultsrelatedtothepresence/absenceofinsertionsequenceISEcp1inthegenomesoftheLebaneseciproﬂoxacin-resistantSalmonellaKentuckystrainsandtheco-localised antimicrobialresistancegenes(ARGs)inthesamecontig.

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Inthisstudy,a93.8-kbCMY-2plasmidregionwasfoundtohave insertedintothechromosomeofSalmonellaKentucky17-70328 (K12)strain.Thechromosomally-integratedIncI1plasmidshowed anunusualIncA/CgeneticenvironmentofblaCMY-2,consistingof

ISEcp1upstreamandblc,

D

sugEand

D

ecnRdownstreamofblaCMY-2.

This conﬁguration seemed to be derived via recombination

betweenIncA/CandIncI1plasmidsthathavecoexistedinancient SalmonellaKentuckyhosts.PreviousreportsonSalmonellaandE. colihaveassignedsuchplasmidinsertionstoanISEcp1-mediated transposition[25,31].Interestingly,weshowedthattheintegrated plasmidresidedbetweentwoIS10,theinsertionsofwhich,asfaras weknow,tookplaceatnoveltargetDNAsites[32].Full-lengthor

partiallyinvertedformsofISEcp1havealreadybeendescribedto co-localisewith IS10 [33]. AnSXT/A391 integrativeconjugative element(ICE)fromProteusmirabilisharbouredasimilar14.2-kb Tn10-like composite transposon containing a truncated ISEcp1 upstreamofblaCMY-2,blc,sugEandecnR[34].However,inourcase,

nofeatures indicativeofICEwereidentiﬁed.Takentogether,all dataprovidedfullevidencethatintegrationofthe93.8-kbCMY-2 fragmentoccurredbyatwo-stepmechanismoftranspositionof IS10coupledtohomologousrecombinationatthetransposition site, and resulting in a cointegrate formation that contains a duplicationofIS10ateachchromosome–plasmidjunction(Fig.4).

Such cointegrate production mechanisms by interplasmid or

Fig.1.SchematicdiagramsshowingthegenomicorganisationoftheISEcp1–blaCMY-2transpositionunitsandﬂankinggenescarriedon(A)twoextrachromosomalIncI1

plasmidvariantsfromfourSalmonellaKentuckystrainsand(B)aplasmidinsertedintothechromosomeofSalmonellaKentuckystrain17-70328(K12).Thechromosomal insertionsitewasidentiﬁedbycomparisonwiththecloseIncI1blaCMY-2SalmonellaKentuckypCVM29188_101plasmidpreviouslycharacterised.Theorientationofeachgene

andISelementisindicatedbyarrows.Blackarrowsrepresentchromosomalopen-readingframes(ORFs)andgreyarrowsrepresentplasmidORFs.TheISEcp1–blaCMY-2

transpositionunitwasdeﬁnedaftertranspositionfromitsoriginalsite,andgenescapturedarerepresentedbymulticolouredarrows.Intergenicredlinesrepresentregions withnoidentitytoIncI1plasmidsusedasascaffold.Invertedrepeats(IRs)ﬂankingthetransposasetnpAgenesarerepresentedbytrianglesasfollows:IRL,leftinvertedrepeat;

IRR,rightinvertedrepeat,IRalt,alternativerightinvertedrepeat.NucleotidesequencesofIRsareshownwithpointmutationswritteninredcharactersandGdeletion

underlined.IS10Ldirectrepeat(DR1)nucleotidesequencesarehighlightedinyellow,whileIS10Rdirectrepeat(DR2)nucleotidesequencesarehighlightedincyanblue. ISEcp1-associated5-bpAT-richdirectrepeats(DR3)arehighlightedinred.TheTGGGTDRsequenceofISEcp1wasdetectedatthesugE5ʹ.TheDR1sequenceoverlapswith9bp ofthehemPORFextendingfrom+2to+10nucleotidepositionswithrespecttothetranslationinitiationsite.TheDR2sequenceoverlapswith9bpoftheSeKA_C0065ORF extendingfrom+16to+24nucleotidepositionswithrespecttothetranslationinitiationsite.Four-pointstarsindicatetruncatedgenesfollowingdeletionevents.H, hypotheticalprotein.Scalesof500bpareshown.

Fig.2. VirulencedeterminantsoftheeightLebaneseSalmonellaKentuckyisolatesbasedontheproteinsequencesoftheSalmonellaspp.database. R.ElHage,C.Losasso,A.Longoetal./JournalofGlobalAntimicrobialResistance23(2020)408–416

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chromosome-to-plasmid transpositions of IS10 have been de-scribedpreviously[35].ItisworthmentioningthatbothIRRofIS10

wereperfectlyduplicatedbutwerenot100%reverse complemen-tary totheir respectiveIRL owingtosome mutations(Fig.1B),

whichcouldaccountfortheirautonomyandstabilitywithinthe chromosome[32].

Genetransferunderantibioticselectivepressurefacilitatesthe spreadofdrugresistance[36].Thiscouldexplainthe dissemina-tionofthehighlyMDRSalmonellaKentuckyST198clonefollowing

theexcessivetherapeuticuseoffluoroquinolones(enrofloxacin), third-generationcephalosporins(ceftiofur)and trimethoprimin theLebanesepoultryindustry.Thesefindingsareinaccordance withotherreportsinAfricaandsomepartsofAsia[6].Inlinewith this,SalmonellaKentuckyiswellknownforitsgenomicplasticity mediatedbyhorizontalacquisitionofplasmidsorgenomicislands

[37]. These include the SGI1-K initially detected in Salmonella KentuckystrainsisolatedinAustralia.ItcomprisesaMDRregionto aminoglycosides[aadA7andaac(3)-Id],tetracyclines[tet(A)]and

Fig.3.Singlenucleotidepolymorphisms(SNP)-basedphylogenetictreeoftheeightLebaneseciproﬂoxacin-resistant(CIPR₎_Salmonella_Kentucky_isolates_with_Salmonella

KentuckyCVM29188asreferencegenome.

Fig.4.Proposedmodelofatwo-stepmechanismoftranspositionfollowedbyintermolecularhomologousrecombinationforintegrationoftheblaCMY-2-carryingIncI1

plasmidintothechromosomeofSalmonellaKentucky17-70328(K12)strain.AchromosomalIS10isexcisedfromthechromosome(donor)andinsertedintotheIncI1 plasmid-borneSeKA_C0065targetgene,causingitsdisruption.RecombinationisassumedtohaveoccurredviatheIS10sequenceitselfsincethesequencesﬂankingIS10inthe chromosomehadnohomologytotheIncI1plasmidasrevealedbywhole-genomesequencing(WGS).AnadditionalcopyoftheIS10sequencemustalwaysexistinclose proximitytothetranspositioncomplexinthechromosome[35],hereinsertedatthechromosomalhemPlocustherebyservingasadonorforhomologousrecombination. BluearrowsindicatetheorientationofIS10elements.OtherprobablesourcesofIS10attheSeKA_C0065locuscouldbethepCVM29188_146plasmid(alreadyknownto harbourIS10),assumedtohavecoexistedwiththeblaCMY-2-carryingIncI1plasmidinaancientbacterialhost,oreventhechromosomeofthisancienthost.

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sulfonamides(sul1)aswellasamercuryresistancemodule,allof which were locatedin a class 1integron [22,38]. Similarly,we showedthatﬁveof eightstrains carriedthis MDRregionalong withtheSGI1-K,exceptforstrain17-70460(K24).Moreover,other non-negligiblecontributorscouldtriggerthismultidrugresistance, suchasfreetradeandtravelaswellastheuseofcontaminated feedsofaquacultureorigininpoultryfarms[5].Fishmealisthe

most common source of mercury for farmed animals [39].

Consequently, selective pressure by mercury in contaminated feeds and/or excessive antibiotic usage in farms directly or indirectlyfavourthemaintenanceofsuchresistancegenesamong Salmonellaisolates.

Virulotyping results revealed little gene variability among sevenSalmonellaKentuckystrains.ThenumberofSPIsvariedfrom onetoﬁveperisolate,withC63P1beingthemostpredominant,as described previously [40]. Most of T3SS-associated geneswere detected inall ourstrainsexceptfor theT3SS-2sspH2gene,as systematicallyreportedforSalmonellaKentucky[41].Thereduced virulenceofSalmonellaKentuckywasthenpartiallyattributedto theabsenceofsspH2[13].TheputativeirontransportersSitABCD andIroNareessentialforSalmonellavirulenceandwereshownto be always present in Salmonella Kentuckyin comparison with many Salmonella serotypes being studied [13]. All our isolates carriedsitC,butsixisolatesharbouredtheiroNgene.Therefore,the presenceofthesegenesinSalmonellaKentuckyisolatesdeserves attentioninpromoting theemergenceofpathogenicSalmonella Kentucky isolates associated with human infection worldwide. Another explanation for Salmonella Kentucky emergence as a predominantcoloniserofthechickencaecummightbethehigh expressionlevelsofRpoS-regulatedgenescomparedwith Salmo-nellaTyphimurium[12].Indeed,this studyhighlightedthehigh conservation of RpoS-regulated genes involved in galactose catabolism and curli production for colonisation of Salmonella Kentuckyinthecaecum.

In conclusion, AmpCβ-lactamase-producing Salmonella Ken-tuckyST198strainsreportedherearetheﬁrstevidenceinLebanon, thereby highlightingtheirhighdissemination inthe

Mediterra-nean Basin. Also, the arsenal of resistance and virulence

determinantsidentifiedinassociationwithmanymobilegenetic elements could strongly promote the emergence of Salmonella Kentucky infectionin humans.Further effortsare neededfrom health,foodandagriculturalauthoritiestocontrolthisemergence. In termsoffoodsafety,ourfindingsrepresentthefirstnational databaseforfuturelegislativeamendmentsthatwillalsoservethe agricultural development policy of the Lebanese Agricultural ResearchInstitute(LARI)andtheMinistryofAgriculture.Inclusion of SalmonellaKentuckyST198 asa targetstrain inany national reductionplanofSalmonellainpoultryisthereforeworthbeing fullyimplemented. Funding None. Competinginterests Nonedeclared. Ethicalapproval Notrequired. Acknowledgments

The authors thank the Istituto Zooproﬁlattico Sperimentale delle Venezie (Italy)for performingthe WGS.The authorsalso

acknowledgeDrMichelAfram(GeneralDirectorofLARI)forhis greatsupport.

AppendixA.Supplementarydata

Supplementary material related to this article can be

found, in the online version, at doi:https://doi.org/10.1016/j. jgar.2020.11.002.

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